The long term objectives are to elucidate the mechanisms involved in the maturation of N-glycans in Saccharomyces cerevisiae, particularly those that have been conserved through eukaryotic evolution. The major specific aims are to study the ER yeast alpha-1,2-mannosidase that trims Man9GlcNAc to a specific isomer of Man8GlcNAc. We have purified and cloned this enzyme, and have obtained high level expression (mg quantities) of the enzymatically active catalytic domain. Structure- function studies of the yeast alpha-1,2-mannosidase will be done, using chemical methods combined with site-directed mutagenesis. The location of disulfide bonds, the role of sulfhydryl groups and the importance of the calcium consensus sequence will be studied. Amino acid residues that participate in catalysis will be identified using active site-directed irreversible inhibitors, and their function will be confirmed by site- directed mutagenesis. Since inhibitors of processing glycosidases have antimetastic and antiviral properties, the yeast processing alpha-1,2- mannosidase serves as a model to understand the mechanism of action of this class of enzymes and may help design more potent inhibitors for therapeutic purposes. By homology with the yeast alpha-1,2-mannosidase, we have also cloned a mouse cDNA encoding a Golgi alpha-1,2-mannosidase that trims Man9GlcNAc to Man5GlcNAc. Both the yeast and mouse enzymes are type II membrane proteins with a homologous C-terminal catalytic domain, but have different N-terminal sequences. The structural determinants responsible for targeting these enzymes to their respective organelles will be studied using yeast/mouse alpha-1,2- mannosidase hybrid molecules for immunolocalization in yeast and mammalian cells. We also plan to purify and clone a novel yeast alpha- 1,2-mannosyltransferase and to determine its function in vivo by gene disruption. Since yeast has advantages in biotechnology for the expression of heterologous glycoproteins with therapeutic potential, these studies are important to develop better strains for the production of mammalian-type carbohydrates.